Assay for tissue factor in a sample

ABSTRACT

The invention provides assays for detecting and quantitating tissue factor and factor VIIa in simple and complex biological systems. The assays are performed by detecting and/or measuring the tissue factor cofactor activity and factor VIIa enzymatic activity using aminonapthalene-based fluorogenic substrates.

This application claims priority from U.S. Provisional Application60/425,662, filed Nov. 6, 2002, and U.S. Provisional Application60/466,214, filed Apr. 28, 2003, each of which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates, generally, to the field of detection of proteinsinvolved in blood coagulation. More specifically, it relates to a methodfor determining the amount of tissue factor and/or factor VIIa in simpleand complex biological systems.

2. Description of the Related Art

Tissue factor (TF) and factor VIIa (fVIIa) are essential components forthe initiation of blood coagulation. Blood coagulation is initiated whencryptic TF becomes exposed on the surface of vascular cells where it canbind circulating fVIIa.

Although several assays for fVIIa have been described (some commerciallyavailable), those assays do not discriminate between factor VII andfactor VIIa, either due to the lack of specificity in immunologicmethods, or due to the feedback-activation of factor VII in theamidolytic and clot-based assays. For example, there is a “direct”factor VIIa assay based upon clotting of plasma initiated with a solublemutant of TF. The assay involves the entire coagulation cascade, so itis therefore sensitive to the concentration of procoagulant proteins andcoagulation inhibitors as well as factor VII by virtue of feed-backactivation. Thus, the clotting time of plasma reflects the concentrationof fVIIa, as well as, the concentration of all components of plasmainvolved in coagulation and its regulation.

Moreover, because of the critical role TF plays in hemostasis, itspotential role in metastasis, and its extensive use in-vitro, it isimportant to have a sensitive and specific TF assay that can detectrelatively low amounts of this protein in biological fluids, cellcultures, lysates, and in purified and semi-purified systems. TF assaysthus far developed employ clotting, chromogenic, and immunochemicalmethods. The clotting methods involve the entire coagulation cascade andare therefore sensitive to alterations in the levels of procoagulantproteins and coagulation inhibitors. Chromogenic methods do not allow adirect measure of TF activity, and are expensive since they requireadditional purified coagulation factors. Similarly, immunochemicalmethods are relatively expensive and time-consuming. Thus, at thepresent time, there is no quick, accurate and somewhat universal methodto directly measure TF activity.

Accordingly, a functional-based assay that could be used to measure TFor fVIIa in purified and/or complex biological systems would have avariety of potential applications. These include: in-vitro diagnosticsfor the assessment of hemostatic potential; in-vitro diagnostics forthrombotic risk assessment; in-vitro diagnostics for cancer screening;quality control during the purification of recombinant tissue factor;quality control during the manufacture of prothrombin time PT reagents;and characterization of final TF and/or PT reagents.

It has been demonstrated that the amidolytic activity of the TF/factorVIIa complex toward small fluorogenic substrates is membrane(phospholipid) independent. This suggests that TF can be successfullyquantitated in a free form in purified systems and biological fluids, aswell as, present on cell or artificial membranes and in cell lysates.Fluorogenic substrates, which allow a quantitation of low concentrationsof factor VIIa (as described above), will similarly allow thequantitation of low concentrations of TF. U.S. Pat. No. 5,399,487, whichis fully incorporated by reference, discloses fluorogenic substrates forserine proteases that contain 6-amino-1-naphthalenesulfonamide (ANSN)leaving groups.

SUMMARY OF INVENTION

The invention provides assays for detecting and quantitating tissuefactor and factor VIIa in purified form or in complex biologicalmixtures such as body fluids and tissues, e.g., plasma. The assay isperformed by detecting and/or measuring the TF-dependent fVIIa enzymaticactivity using aminonapthalene sulfonamide-based (ANSN-based)fluorogenic substrates. The TF-dependent activity is an important aspectof this assay, as the TF/factor VIIa complex will yield nearly a100-fold higher rate of substrate hydrolysis relative to factor VIIaalone with appropriate substrates. It has been demonstrated that theproperties of the ANSN-based fluorogenic substrates allow for the directquantitation of fVIIa at low (<5) picomolar concentrations in purifiedsystems.

The enzymatic activity of a TF/fVIIa complex is related to theconcentration of either TF or fVIIa in the sample, depending upon whichis present at a limiting concentration. Standard calibration curves canbe generated using samples with known concentrations of TF or fVIIa. Theconcentration of active TF or fVIIa in a sample suspected of containingthese factors can be determined by comparing the detected enzymaticactivity to the calibration curves.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a fluorogenic assay calibration curve for factor VIIaamidolyic activity in the presence of excess (5 nM) TF;

FIG. 2 is a fluorogenic assay calibration curve for TF cofactor activityin the presence of excess (2 nM) factor VIIa; and

FIG. 3 is a calibration curve for TF activity where TF activity isexpressed as the amount of cofactor activity per milliliter of solution(units/ml). RFU represents relative fluorescent units.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the invention provides assays for tissue factor andfactor VIIa. There are two broad aspects of the invention.

One aspect is a method used to determine the cofactor activity of TF ina fluid sample suspected of containing TF or containing an unknownquantity of TF. The term “determine” as used herein encompasses bothquantitative measurements as well as qualitative assessments. Thus, themethods of the invention can be used to determine (1) if any TF ispresent in a sample and (2) the amount of TF activity in a sample. Inthe first aspect, the assay initially involves combining the samplesuspected to contain TF with fVIIa to form a TF/fVIIa complex, i.e., areaction mixture. Optionally, added to the reaction mixture are divalentmetal ions, such as calcium ion, or metal ion chelators, such asethylenediamimetetraacetic acid (EDTA) and ethyleneglycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA). Otheroptional components of the reaction mixture include alcohols andpolyhydroxylated materials. Representative alcohols include loweralcohols, e.g., C₁-C₆ alcohols such as methanol, ethanol, propanol,pentanol, and hexanol. Polyhydroxylated materials include variousglycols and sugars. Representative polyhydroxylated materials includeglycerol, ethylene glycol, diethylene glycol, triethylene glycol,propylene glycol, dipropylene glycol, trimethylene glycol, butyleneglycols, 1,2-cyclohexanediol, poly(oxyalkylene)polyols derived from thecondensation of ethylene oxide, propylene oxide, or any combinationthereof, 1,1,1-trimethylolpropane, 1,1,1-trimethylolethane,2,2-dimethyl-1,3-propane diol, and pentaerythritol. More preferably, thealcohol is ethanol or n-propanol. Preferred polyhydroxylated materialsinclude glycerol, ethylene glycol, and propylene glycol. Morepreferably, the polyhydroxylated material is ethylene glycol orpropylene glycol.

Typically, the TF sample is combined with a molar excess of fVIIa toproduce a TF/fVIIa enzyme complex in a TF limiting manner. Whendeveloping a calibration curve, there is a possibility that fVIIa maynot be present in an excess relative to the concentration of TF. If so,there will be no change in the fluorescence level with change inconcentration, i.e., a flat line will be generated, until the TFconcentration is reduced to a point where fVIIa is in excess.Preferably, the excess of fVIIa is about a 2-fold excess. Alternatively,the molar excess of fVIIa is about a 100-fold molar excess; in yetanother alternative, the excess of fVIIa is about a 1000-fold excess.Finally, the enzymatic activity of the complex may be detected,preferably by using an amino-napthalenesulfonamide-based fluorogenicsubstrate. Fluorescence can be monitored (continuously ordiscontinuously) using a suitable fluorescence spectrophotometer.Representative devices include (a) Perkin Elmer model MPF-44A; (b) aPerkin Elmer Model LS50B; (c) a Molecular Devices Spectramaxfluorescence plate reader. When an ANSN-based fluorescent substrate isused, the fluorophore is preferably detected using monochrometers set atan excitation wavelength of about 340-360 nm, preferably about 350 nm,and an emission wavelength of about 460-480 nm, preferably about 470 nm.Light scattering artifacts can be minimized using an appropriate cut-offfilter in the emission light beam, e.g., a 435 nm cut off filter.

The amino-naphthalenesulfonamide (ANSN) based fluorogenic substrate usedis a compound of the formula:

or a pharmaceutically acceptable non-toxic salts thereof; wherein

-   -   R₁ is hydrogen, straight or branched chain lower alkyl having        1-6 carbon atoms optionally substituted with C₁-C₆ alkoxy,        straight or branched chain alkenyl having 2-8 carbon atoms,        straight or branched chain alkynyl having 2-8 carbon atoms,        cycloalkyl having 3-7 carbon atoms, alkylcycloalkyl where the        alkyl portion has 1-6 carbon atoms, cycloalkylakyl where the        alkyl portion has 1-6 carbon atoms, or phenylalkyl where the        alkyl portion is straight or branched chain alkyl having 1-6        carbon atoms, or a group of the formula

-   -   -   R₅ represents hydrogen or an amino acid side chain; and

    -   R₄ is hydroxy, C₁-C₆ alkoxy, an amino acid or a peptide residue;        -   R₂ is hydrogen, straight or branched chain lower alkyl            having 1-6 carbon atoms, straight or branched chain alkenyl            having 2-8 carbon atoms, straight or branched chain alkynyl            having 2-8 carbon atoms, cycloalkyl having 3-7 carbon atoms,            alkylcycloalkyl where the alkyl portion has 1-6 carbon            atoms, or phenylalkyl where the alkyl portion is straight or            branched chain alkyl having 1-6 carbon atoms, or a group of            the formula

-   -   -   R₅ represents hydrogen or an amino acid side chain; and        -   R₄ is hydroxy, C₁-C₆ alkoxy, an amino acid or peptide            residue; or

    -   NR₁R₂ forms a nitrogen heterocycle; and

    -   R₃ is an amino acid or a peptide residue.

These substrates can be prepared as described in U.S. Pat. No.5,399,437. Suitable substrates include the following:D-FPR-(cyclohexyl)ANSN (where FPR represents Phe-Pro-Arg, ANSNrepresents aminonaphthalenesulfonamide, R₁ is cyclohexyl and R₂ ishydrogen); D-FPR-(ethyl)ANSN (R₁ is ethyl and R₂ is hydrogen),D-FPR-(n-propyl)ANSN (R₁ is n-propyl and R₂ is hydrogen),D-FPR-(n-butyl)ANSN (R₁ is n-butyl and R₂ is hydrogen),D-FPR-(n-hexyl)ANSN (R₁ is n-hexyl and R₂ is hydrogen),D-FPR-(benzyl)ANSN (R₁ is benzyl and R₂ is hydrogen),D-FPR-(hexamethylene)ANSN (NR₁R₂ represents an azepan-1-yl group),D-FPR-(isopropyl)ANSN (R₁ is isopropyl and R₂ is hydrogen),D-FPR-(methoxyethylene)ANSN (R₁ is methoxyethyl and R₂ is hydrogen),D-FPR-(t-butyl)ANSN (R₁ is t-butyl and R₂ is hydrogen),D-FPR-(methylacetate)ANSN (R₁ is —CH₂CO₂CH₃ and R₂ is hydrogen),D-FPR-(di-ethyl)ANSN (R₁ and R₂ are both ethyl),Boc-D-FPR-(cyclohexyl)ANSN (R₁ is cyclohexyl and R₂ is hydrogen),(p-F)FPR-(ethyl)ANSN (R₁ is ethyl and R₂ is hydrogen),Boc(p-F)FPR-(ethyl)ANSN (R₁ is ethyl and R₂ is hydrogen),D-FVR-(ethyl)ANSN (R₁ is ethyl and R₂ is hydrogen),Boc-D-FVR-(ethyl)ANSN (R₁ is ethyl and R₂ is hydrogen),D-LPR-(propyl)ANSN (R₁ is propyl and R₂ is hydrogen),Boc-D-LPR-(propyl)ANSN (R₁ is propyl and R₂ is hydrogen),D-VPR-(butyl)ANSN (R₁ is n-butyl and R₂ is hydrogen),Boc-D-VPR-(butyl)ANSN (R₁ is n-butyl and R₂ is hydrogen),L-VPR-(butyl)ANSN (R₁ is n-butyl and R₂ is hydrogen),Boc-L-VPR-(butyl)ANSN (R₁ is n-butyl and R₂ is hydrogen),D-VLR-(butyl)ANSN (R₁ is n-butyl and R₂ is hydrogen),Boc-D-VLR-(butyl)ANSN (R₁ is n-butyl and R₂ is hydrogen),L-VLR-(butyl)ANSN (R₁ is n-butyl and R₂ is hydrogen),Boc-L-VLR-(butyl)ANSN (R₁ is n-butyl and R₂ is hydrogen),D-LSR-(propyl)ANSN (R₁ is propyl and R₂ is hydrogen),Boc-D-LSR-(propyl)ANSN (R₁ is propyl and R₂ is hydrogen),D-FLR-(propyl)ANSN (R₁ is propyl and R₂ is hydrogen),Boc-D-FLR-(propyl)ANSN (R₁ is propyl and R₂ is hydrogen),L-FLR-(propyl)ANSN (R₁ is propyl and R₂ is hydrogen),D-VSR-(isopropyl)ANSN (R₁ is isopropyl and R₂ is hydrogen),Boc-D-VSR-(isopropyl)ANSN (R₁ is isopropyl and R₂ is hydrogen),D-LGR-(cyclohexyl)ANSN (R₁ is cyclohexyl and R₂ is hydrogen),Boc-D-LGR-(cyclohexyl)ANSN (R₁ is cyclohexyl and R₂ is hydrogen),D-PFR-(isopropyl)ANSN (R₁ is isopropyl and R₂ is hydrogen), andMes-D-LGR(di-ethyl)ANSN (R₁ and R₂ are both ethyl). A preferredsubstrate is D-FPR-(cyclohexyl)ANSN.

In addition to fluorogenic substrates, chromogenic substrates such asp-nitroaniline-based (pNA-based) substrates may be employed.

Tissue factor from a variety of sources and species can be assayed usingthe invention. Preferably, the TF to be assayed with the invention ishuman TF. There are several sources for human TF. The sources includebrain tissue, placenta, endothelial cells, tissue extract, plasma, cellextract, synthetic or naturally derived thromboplastin, and recombinanthuman TF. The fVIIa used in the TF assay is preferably either nativehuman factor VIIa or recombinant factor VIIa, although factor VIIa fromother species and sources may be employed.

The concentration of TF in the sample can be determined by quantifyingthe TF-dependent enzymatic activity of the TF/fVIIa complex. Thisinvolves comparing the TF-dependent enzymatic activity to a standardcalibration curve. Quantifying the TF-dependent enzymatic activity ofthe TF/fVIIa complex in a sample with known concentrations of TFgenerates the standard curve. The concentrations of TF used for creatingthe standard curve may range from about 0.1 pM to about 1 mM, as long asfactor VIIa is maintained in a molar concentration greater than that ofTF, i.e., in excess relative to the TF molar concentration.

The specificity, sensitivity and limits of detection of the tissuefactor assay may be modulated by employing techniques to physicallycapture tissue factor from the sample solution. This may be accomplishedby, for example, using immunocapture techniques that employ immobilizedanti-tissue factor antibodies or by immobilizing the enzyme (factorVIIa) itself. Using such techniques, tissue factor can be captured orremoved from solution while extraneous materials are washed away.Techniques suitable for adhering antibodies to assay plates arewell-known in the art as are methods for immobilizing enzymes such asfVIIa. Suitable anti-tissue factor antibodies are commercially availableor may be prepared using methods known in the art.

The performance of the tissue factor assay can be enhanced if desired byadjusting various assay conditions. For example, the pH and ionicstrength of the assay buffer can be adjusted.

The second aspect of the invention is a method used to determine thefVIIa enzymatic activity in a sample, preferably a fluid sample,suspected of containing fVIIa or containing an unknown quantity offVIIa.

As noted above, the term “determine” as used herein encompasses bothquantitative measurements as well as qualitative assessments. Thus, themethods of the invention can be used to determine (1) if any fVIIa ispresent in a sample and (2) the amount of fVIIa activity in a sample.

The assay in the second aspect first involves combining TF and fVIIa toform a TF/fVIIa complex, i.e., a reaction mixture. Optionally, added tothe reaction mixture are divalent metal ions, such as calcium ion, ormetal ion chelators, such as ethylenediamimetetraacetic acid (EDTA) andethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid(EGTA). Other optional components of the reaction mixture includealcohols and polyhydroxylated materials. Representative alcohols includelower alcohols, e.g., C₁-C₆ alcohols such as methanol, ethanol,propanol, pentanol, and hexanol. Polyhydroxylated materials includevarious glycols and sugars. Representative polyhydroxylated materialsinclude glycerol, ethylene glycol, diethylene glycol, triethyleneglycol, propylene glycol, dipropylene glycol, trimethylene glycol,butylene glycols, 1,2-cyclohexanediol, poly(oxyalkylene)polyols derivedfrom the condensation of ethylene oxide, propylene oxide, or anycombination thereof, 1,1,1-trimethylolpropane, 1,1,1-trimethylolethane,2,2-dimethyl-1,3-propane diol, and pentaerythritol. More preferably, thealcohol is ethanol or n-propanol. Preferred polyhydroxylated materialsinclude glycerol, ethylene glycol, and propylene glycol. Morepreferably, the polyhydroxylated material is ethylene glycol orpropylene glycol.

Factor VIIa from a variety of sources and species can be assayed usingthe methods of the invention. Preferably, the fVIIa to be assayed ishuman factor VIIa. Possible sources include plasma, tissue extract, cellextract or recombinant material. Preferably, the TF used in the fVIIaassay is native human tissue factor or recombinant human tissue factor,although TF from other sources may be employed. Possible sources of TFalso include synthetic or naturally derived thromboplastin.

The concentration of fVIIa in the sample can be found by quantifying thefVIIa-dependent enzymatic activity of the TF/fVIIa complex. Thisinvolves comparing the fVIIa dependent enzymatic activity to a standardcalibration curve. Quantifying the fVIIa-dependent enzymatic activity ofthe TF/fVIIa complex in a sample with known concentrations of fVIIagenerates the standard curve. The concentrations of fVIIa used forcreating the standard curve may range from about 0.1 pM to about 1 mM,so long as TF is maintained in a molar excess over the factor VIIa.

When developing a calibration curve, there is a possibility that TF maynot be present in an excess relative to the concentration of fVIIa. Ifso, there will be no change in the fluorescence level with change inconcentration, i.e., a flat line will be generated, until the fVIIaconcentration is reduced to a point where TF is in excess. Preferably,the excess of TF is about a 2-fold excess; alternatively, the molarexcess of TF is about a 100-fold molar excess; in another alternative,the excess of TF is about a 1000-fold excess.

Enzymatic activity of the complex may be determined in a manner similarto that used to assay for TF.

The specificity, sensitivity and limits of detection of the basic factorVIIa assay described above may be enhanced by employing techniques tophysically capture factor VIIa from the sample solution. This may beaccomplished using immunocapture techniques that employ immobilizedanti-factor VIIa antibodies or by immobilizing the cofactor (tissuefactor) itself. In this manner, factor VIIa may be captured fromsolution while extraneous materials are washed away in subsequent steps

The performance of the factor VIIa assay can be enhanced if desired byadjusting various assay conditions. As with the TF assay, for example,the pH and ionic strength of the assay buffer can be adjusted.

This invention is illustrated further by the following examples, whichare not to be construed as limiting the invention in scope or spirit tothe specific compounds or procedures described in them.

Example 1

Varying concentrations of fVIIa (0-1000 pM) were incubated with 5 nM TFin 20 mM Hepes (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid),0.15 M Sodium Chloride (NaCl), pH 7.4 (HBS), containing 20 mM EDTA for10 minutes at room temperature, followed by the addition of 100 μMD-FPR-(cyclohexyl)ANSN. Substrate hydrolysis was monitored continuouslyfor five minutes in a fluorometer at an excitation wavelength of 350 nmand an emission wavelength of 470 nm. The rates of substrate hydrolysiswere determined for each fVIIa concentration tested. FIG. 1 is a graphof a calibration line based on these rates of hydrolysis at variousfactor VIIa concentrations. FIG. 1 shows that the factor VIIa/TFdependent rate of substrate hydrolysis is linear over the range offactor VIIa concentrations tested.

Example 2

Normal citrated plasma was diluted (1:1) with HBS containing 20 mM EDTA,and the pH was adjusted to 7.4. Five nM TF was then added. The plasmawas incubated for 10 minutes at room temperature followed by theaddition of 100 μM D-FPR-(cyclohexyl)ANSN. The rates of substratehydrolysis were determined. In a control experiment, the rate ofsubstrate hydrolysis was evaluated for the same plasma sample, but inthe absence of TF. The increased rate of substrate hydrolysis observedin the presence of TF (versus the control) was attributed to theactivity of the factor VIIa/TF complex. The results were compared to theFIG. 1 calibration curve. The plasma sample yielded a fVIIaconcentration of 102 pM.

Example 3

Concentrations of TF were varied (0-500 pM) and incubated with 2 nMfVIIa in HBS pH 7.4, containing 2 mM Calcium Chloride (CaCl₂), for 10minutes at room temperature. Next, 50 μM D-FPR-(n-butyl)ANSN was added.Substrate hydrolysis was then monitored continuously for five minutes ina fluorometer at an excitation wavelength of 350 nm and an emissionwavelength of 470 nm. The rates of substrate hydrolysis were determinedfor each TF concentration tested. The rate of hydrolysis by 2 nM fVIIaalone was measured and subtracted from the rates observed in thepresence of TF. FIG. 2 is a graph of a calibration line based on theserates of hydrolysis at various TF concentrations. FIG. 2 shows that thefactor VIIa/TF dependent rate of substrate hydrolysis is linear over therange of TF concentrations tested.

Example 4

In this example the preparation for TF employed in Example 3 wasrelipidated into phospholipid (PCPS) vesicles, composed of 75%phosphatidylcholine (PC) and 25% phosphatidylserine (PS). RelipidatedTF, 250 pM TF to 500 nM PCPS, was incubated with 2 nM fVIIa in HBS, 2 mMCaCl₂ pH 7.4 for ten minutes at room temperature. Next, 50 μMD-FPR-(n-butyl)ANSN was added. Rates of hydrolysis were determined andthe rate of hydrolysis by 2 nM fVIIa alone was measured and subtractedfrom the rates observed in the presence of TF. The net rate ofhydrolysis was then compared to the FIG. 2 calibration curve. The assaydata indicates that 35-40% of the TF added to the relipidation mixturewas expressed as functional TF, which indicates poor recovery of totalTF activity following relipidation.

Example 5

A standardized preparation of recombinant human TF with a cofactoractivity of 241 units/ml (specific activity of 3906 units/milligram) wasserially diluted to create stock assay standards in the range of 8 to0.25 units/ml. One hundred microliters of each stock standard wascombined with 50 microliters of 120 nM factor VIIa. Subsequently 50microliters of 150 uM D-FPR-(n-butyl)ANSN was added and the rate ofsubstrate hydrolysis was measured in a fluorometer at an excitationwavelength of 350 nm and an emission wavelength of 470 nm. A standardcurve was generated in FIG. 3 by plotting the change in fluorescentintensity over time versus the concentration of TF (in units/ml). In asimilar manner, a sample of TF (0.8 mg/ml) with an unknown amount ofcofactor activity was assayed for comparison to the standard. Aftercorrecting for assay dilution, the unknown sample returned an assayvalue of 2803 units per milliliter, and thus a specific activity of 3504units per milligram. Consequently, this allows for comparison ofdifferent preparations of TF using their specific activity, which is adirect indication of quality and functionality.

Example 6

Varying concentrations of fVIIa (0-200 pM) were incubated with 40 nM nMTF, in a buffer of 20 mM Hepes(N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid), 0.05 M SodiumChloride (NaCl), pH 8.0 (HBS), containing 0.1% (w/v) polyethlyeneglycol(PEG) and 25% (v/v) ethylene glycol for 10 minutes at room temperature,followed by the addition of 80 μM D-FPR-(cyclohexyl)ANSN. Substratehydrolysis was allowed to proceed for 24 hours at room temperature afterwhich an endpoint reading was taken in a fluorometer at an excitationwavelength of 350 nm and an emission wavelength of 470 nm. When acalibration curve was constructed by plotting the relative fluorescenceintensity versus factor VIIa concentration, a linear relationship wasobserved with the lower limit of detection being approximately 2 pM. Inthis case, the optimized buffer conditions (pH, ionic strength, andadditives) along with an extended assay period and end-point readingyielded a lower limit of detection that was 20 fold lower than thatobserved using the standard assay conditions outlined in “example 1”.

Example 7

A microtiter assay plate was precoated with purified recombinant tissuefactor and residual plate binding sites were blocked with bovine serumalbumin using conventional coating and blocking methods. Serialdilutions of factor VIIa in TBS, pH 7.4 (20 nM to 8.5 pM) were added tothe plate and allowed to incubate for 2 hours at room temperature. Theplate was then washed with TBS, pH 7.4 to remove non-bound factor VIIaand this was followed by the addition of 80 μM D-FPR-(cyclohexyl)ANSN.Substrate hydrolysis was allowed to proceed for 24 hours at roomtemperature after which an endpoint reading was taken in a fluorometerat an excitation wavelength of 350 nm and an emission wavelength of 470nm. When a calibration curve was constructed by plotting the relativefluorescence intensity versus factor VIIa concentration, a linearrelationship was observed with the lower limit of detection beingapproximately 8.5 pM.

The invention and the manner and process of making and using it, are nowdescribed in such full, clear, concise and exact terms as to enable anyperson skilled in the art to which it pertains, to make and use thesame. It is to be understood that the foregoing describes preferredembodiments of the present invention and that modifications may be madetherein without departing from the spirit or scope of the invention asset forth in the claims. To particularly point out and distinctly claimthe subject matter regarded as invention, the following claims concludethis specification.

1. A method for determining the concentration of tissue factor (TF) in asample suspected to contain TF, comprising: (a) combining the sample anda molar excess of factor VIIa (fVIIa) compared to the moles of TF in thesample to produce a TF/fVIIa enzyme complex; (b) detecting the enzymaticactivity of the complex using a fluorogenic or chromogenic substrate;(c) generating numerical values correlated with the enzymatic activityof the sample; and (d) comparing the numerical values with a standardcurve of TF-dependent enzymatic activity, wherein the standard curve isgenerated by quantifying TF-dependent enzymatic activity of the TF/fVIIacomplex in samples with known concentrations of TF.
 2. The method ofclaim 1, wherein the substrate is a compound of the formula:

or a pharmaceutically acceptable non-toxic salts thereof; wherein R₁ ishydrogen, straight or branched chain lower alkyl having 1-6 carbon atomsoptionally substituted with C₁-C₆ alkoxy, straight or branched chainalkenyl having 2-8 carbon atoms, straight or branched chain alkynylhaving 2-8 carbon atoms, cycloalkyl having 3-7 carbon atoms,alkylcycloalkyl where the alkyl portion has 1-6 carbon atoms,cycloalkylalkyl where the alkyl portion has 1-6 carbon atoms, orphenylalkyl where the alkyl portion is straight or branched chain alkylhaving 1-6 carbon atoms, or a group of the formula:

R₅ represents hydrogen or an amino acid side chain; and R₄ is hydroxy,C1-C6 alkoxy, an amino acid or a peptide residue; R₂ is hydrogen,straight or branched chain lower alkyl having 1-6 carbon atoms, straightor branched chain alkenyl having 2-8 carbon atoms, straight or branchedchain alkynyl having 2-8 carbon atoms, cycloalkyl having 3-7 carbonatoms, alkylcycloalkyl where the alkyl portion has 1-6 carbon atoms, orphenylalkyl where the alkyl portion is straight or branched chain alkylhaving 1-6 carbon atoms, or a group of the formula:

R₅ represents hydrogen or an amino acid side chain; and R₄ is hydroxy,Cl-C6 alkoxy, an amino acid or peptide residue; or NR₁R₂ forms anitrogen heterocycle; and R₃ is an amino acid or a peptide residue. 3.The method of claim 1, where the substrate is a chromogenic substrate.4. The method of claim 3, wherein the chromogenic substrate is apara-nitroaniline based substrate.
 5. The method of claim 1, wherein theTF is native human tissue factor.
 6. The method of claim 1, wherein thesample is obtained from is brain tissue, placenta, endothelial cells,tissue extract, plasma, cell extract, synthetic or naturally derivedthromboplastin, or recombinant human tissue factor.
 7. The method ofclaim 1, wherein the fVIIa is native human factor VIIa or recombinantfactor VIIa.
 8. The method of claim 1, wherein the TF and the fVIIa arenot of human origin.
 9. The method of claim 1, wherein the concentrationof TF is from 0.1 pM to 1 mM.
 10. The method of claim 1, wherein thereaction mixture contains divalent metal ion or a metal ion chelator.11. The method of claim 10, wherein the divalent metal ion is calciumion, magnesium ion or manganese ion.
 12. The method of claim 10, whereinthe metal ion chelator is ethylenediamimetetraacetic acid (EDTA) orethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid(EGTA).